Technical Field
The present invention relates to a method for examining microorganisms, and in particular, relates to a method for examining microorganisms, the method being suitable for detecting microorganisms such as planktons that are included and live in ballast water or the like.
Background Art
A ship carrying no cargoes is loaded with ballast water in order to stabilize the ship when it sails, and discharges the ballast water in a marine area where cargoes are carried on the ship.
The ballast water is usually discharged in a marine area different from the marine area where the ballast water is loaded on the ship, and therefore, the following problem may be caused: microorganisms such as planktons and bacteria included in the ballast water are transported to a marine area other than the native habitats thereof to disrupt ecosystem.
In order to address such a problem, international rules for the regulation of ballast water have been established, and “the International Convention for the Control and Management of Ships' Ballast Water and Sediments (Ballast Water Management Convention)” has been adopted.
In the “Guidelines for Ballast Water Sampling (G2)” related to the above Ballast Water Management Convention, “Ballast Water Discharge Standards (D-2)” prescribes the acceptable population of microorganisms that are included and live in ballast water discharged from a ship with differentiation based on the minimum size of the microorganisms, and for example, it prescribes that the acceptable population of microorganisms having a minimum size of 50 μm or more (hereinafter, referred to as “L size organisms”) is 10/m3 or less, and that of microorganisms having a minimum size of 10 μm or more and less than 50 μm (hereinafter, referred to as “S size organisms”) is 10/mL or less.
As a technique for confirming whether the above Discharge Standards are satisfied when the ballast water is discharged, examination apparatuses for microorganisms have been heretofore known, such as an examination apparatus described in Patent Literature 1, in which seawater pumped up by a water pump is passed through flow cells and subjected to image measurement, and an examination apparatus described in Patent Literature 2, in which seawater pumped up by a water pump is passed through a unit of filters having different apertures, followed by allowing microorganisms on the filters to emit light and counting the number of the microorganism.
The examination apparatus for microorganisms described in Patent Literature 1 includes a staining portion that, while allowing a liquid analyte to flow, stains organisms having living cells present in the analyte; a concentrating portion that, while allowing the stained analyte to flow, performs concentrating so that the concentration of the organisms is increased; an individual measurement portion that acquires image information on individuals including the organisms in the concentrated analyte; and a controlling means that performs measurement of the organisms based on the image information on the individuals output by the individual measurement portion.
Thus, the examination apparatus can perform a step of staining the organisms in the liquid of the analyte, a step of concentrating the organisms in the liquid, a step of acquiring the information on the organisms in the liquid, and the like in a flow system, and therefore has the following advantages as compared with a technique in which the each steps are performed in a batch system: a waiting time until the analyte after completion of one step partially proceeds to the next step can be significantly reduced or can be eliminated, and stable information on the life and death of the organisms can be acquired in the view that deterioration in the state of staining during the waiting time is prevented.
The examination apparatus for microorganisms described in Patent Literature 1, however, allows the seawater pumped by the water pump to sequentially pass through the each steps, and therefore has the problems of being a large-scale apparatus and being high in production cost. Further, although the examination apparatus allows the seawater to sequentially pass through the each steps for a reduction in waiting time, it has the problem of taking at least several hours for the completion of measurement.
In addition, in the examination apparatus for microorganisms described in Patent Literature 2, there are a step of passing the seawater through the unit of filters which is formed by arranging of three filters each having a different aperture in series; a step of allowing the microorganisms trapped in the filters and living therein to conduct any of color development, light emission and fluorescence emission; and a step of detecting any of color development, light emission and fluorescence emission to count the number of the microorganisms in the ballast water or seawater by image analysis.
Thus, the following advantage is provided: capturing the microorganisms by the stepwise size can be realized and, as a result, whether the standard of the acceptable residue for each size is satisfied can be rapidly measured.
The examination apparatus for microorganisms described in Patent Literature 2, however, also allows the seawater pumped by the water pump to sequentially pass through the each steps, and has the problems of being a large-scale apparatus and being high in production cost, as in the examination apparatus for microorganisms described in Patent Literature 1.
In view of the above problems, the present applicant has proposed, in Patent Literature 3, a method for examining microorganisms, in which a batch type measurement cell can be utilized to thereby measure the number of microorganisms in ballast water simply in a short time at a high accuracy.
The method for examining microorganisms proposed by the present applicant includes a stirring and mixing step of stirring and mixing a sample solution obtained by adding a fluorescence staining reagent to a sample in a batch type sample container; an excitation step of irradiating a surface to be irradiated of the sample container with excitation light while stirring the sample solution; a light reception step of counting the number of fluorescence emissions of microorganisms that emit fluorescence by the excitation light; and an estimation step of the number of microorganisms, which is a step of calculating the amount of microorganisms included in the sample in the sample container from the number of the fluorescence emissions detected in the light reception step.
Therefore, the method has the following actions and effects: microorganisms can brightly emit light in an extremely short time, by which the amount the microorganisms in the ballast water can be measured simply in a short time; and the thickness portion of the fluorescence emission is reduced, which results in that the difference in the amount of light between the background and the fluorescence emission of microorganisms become extremely clear to enhance the detection accuracy of the fluorescence emission of microorganisms.
For example, as illustrated in FIG. 6, the detection principle of microorganisms described above is as follows: microorganisms such as planktons subjected to fluorescence staining can be detected as electric signals in a photomultiplier tube, and a background component, for example, of a voltage of about 0.9 V continuously detected can be compared with a fluorescence intensity when living planktons pass to distinguish the peak height of the fluorescence intensity when the planktons pass, namely, the height of the voltage from the background component. The background component here is based on the fluorescence of sample water by itself, namely, intrinsic fluorescence or the fluorescence due to spontaneous decomposition of a staining agent in sample water.
In the above proposition, however, as the background component is larger, a noise component tends to be also larger, and as a result, a problem is that a larger background component causes a reduction in the S/N ratio to affect the detection accuracy.